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Management of acquired resistance to EGFR TKI–targeted therapy in advanced non-small cell lung cancer

Management of acquired resistance to EGFR TKI–targeted therapy in advanced non-small cell lung... Recent advances in diagnosis and treatment are enabling a more targeted approach to treating lung cancers. Therapy targeting the specific oncogenic driver mutation could inhibit tumor progression and provide a favorable prognosis in clinical practice. Activating mutations of epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) are a favorable predictive factor for EGFR tyrosine kinase inhibitors (TKIs) treatment. For lung cancer patients with EGFR-exon 19 deletions or an exon 21 Leu858Arg mutation, the standard first-line treatment is first- generation (gefitinib, erlotinib), or second-generation (afatinib) TKIs. EGFR TKIs improve response rates, time to progression, and overall survival. Unfortunately, patients with EGFR mutant lung cancer develop disease progression after a median of 10 to 14 months on EGFR TKI. Different mechanisms of acquired resistance to first-generation and second-generation EGFR TKIs have been reported. Optimal treatment for the various mechanisms of acquired resistance is not yet clearly defined, except for the T790M mutation. Repeated tissue biopsy is important to explore resistance mechanisms, but it has limitations and risks. Liquid biopsy is a valid alternative to tissue re-biopsy. Osimertinib has been approved for patients with T790M-positive NSCLC with acquired resistance to EGFR TKI. For other TKI-resistant mechanisms, combination therapy may be considered. In addition, the use of immunotherapy in lung cancer treatment has evolved rapidly. Understanding and clarifying the biology of the resistance mechanisms of EGFR-mutant NSCLC could guide future drug development, leading to more precise therapy and advances in treatment. Background growth factor receptor (EGFR) mutation-positive patients. In the United States, an additional 224,390 new lung cancer EGFR mutation-positive patients with lung adenocarcin- cases were diagnosed in 2016, and accounted for about 27% oma had aresponserateas highas 80%,and around 10– of all cancer deaths [1]. Although standard platinum-based 14 months of progression-free survival (PFS) [5, 6]. The chemotherapy is the cornerstone of systemic therapy, it has American Society of Clinical Oncology (ASCO), European amodesteffectonoverall survival (OS) [2]. Lung cancer is Society for Medical Oncology (ESMO) and National still the leading cause of cancer death worldwide [3]. Comprehensive Cancer Network (NCCN) guidelines rec- In the most recent decade, treatment of non-small cell ommend EGFR TKIs as first-line treatment for EGFR-mu- lung cancer (NSCLC) has evolved to a great extent. The tant patients. The most common activating mutations are discovery of driver mutations in lung cancer allows the cre- in-frame deletions in exon 19 and single-point mutation of ation of personalized targeted treatment. It is important exon 21 (Leu858Arg), which together account for more that lung cancer patients are tested for oncogenic drivers of than 80% of known activating EGFR mutations [7, 8]. cancer and receive matched targeted therapy [4]. Epidermal Although EGFR TKIs have a favorable and durable treat- growth factor receptor tyrosine kinase inhibitors (EGFR ment response, most patients will eventually develop pro- TKIs) provide a favorable treatment outcome in epidermal gressive disease (PD) within about one year of treatment. Furthermore, acquired resistance develops and limits the long-term efficacy of these EGFR TKIs. A variety of mecha- * Correspondence: jyshih@ntu.edu.tw nisms of acquired resistance to EGFR TKIs have been Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 100, Taiwan © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wu and Shih Molecular Cancer (2018) 17:38 Page 2 of 14 reported. The most common mechanism is the develop- previously beneficial EGFR TKI, it maybereasonabletocon- ment of acquired EGFR T790M mutation [9]. T790M was sider local treatment and continuation of the targeted agent. found in about 50% of EGFR–mutant cases that acquired This approach yielded more than six months of additional resistance to EGFR TKIs [9]. Patients using either first- or disease control [24, 25]. second-generation EGFR TKIs had a similar prevalence of Yang et al. proposed another criteria for EGFR TKI failure acquired T790M [10]. modes in NSCLC [26]. Based on the duration of disease Preclinical data showed that the second-generation control, the evolution of the tumor burden, and clinical EGFR TKI, afatinib, could overcome the resistance caused symptoms, regardless of genotype profile, the diversity of by the T790M mutation [11], but clinical trials have not EGFR TKI failure could be categorized into three modes, revealed the effect due to toxicity limitations. The narrow including dramatic progression, gradual progression, and therapeutic window of afatinib caused severe adverse ef- local progression. The median PFS was 9.3, 12.9, and fects (AEs), probably owing to inhibition of wild-type 9.2 months (p = 0.007) for these three modes, respectively, EGFR [12, 13]. In the phase III LUX-Head & Neck 1 and median OS was 17.7, 39.4, and 23.1 months (p < 0.001), (LHN1) trial, second-line afatinib significantly improved respectively. In patients with disease in the gradual progres- PFS versus methotrexate in patients with recurrent/meta- sion mode, continuing EGFR TKI therapy was superior to static head and neck squamous cell carcinoma [14]. This switching to chemotherapy in terms of OS (39.4 vs. suggests afatinib is a drug active against wild-type EGFR. 17.8 months; p = 0.02) [26]. Determination of the clinical The third-generation EGFR TKI, osimertinib, has been mode could favor strategies for subsequent treatment and approved for patients with T790M-positive NSCLC with prediction of survival. acquired resistance to EGFR TKIs. Use of third-generation EGFR TKIs was related to different acquired resistance Mechanisms of acquired resistance to EGFR TKIs mechanisms [15–18]. Therefore, in this manuscript, we Acquired resistance mechanisms vary. Several study groups focused on these recently developed treatment strategies comprehensively explored the mechanisms through re- for EGFR-mutant NSCLC with acquired resistance to biopsy tissue specimens. The most common acquired first- or second-generation EGFR TKIs. resistance mechanisms were of three types: target gene modification, alternative pathway activation and histological Clinical presentation of acquired resistance to first-line or phenotypic transformation (Fig. 1). EGFR TKIs Although EGFR-mutant patients receiving EGFR TKIs Target gene modification have longer median PFS than those receiving platinum- The T790M mutation, which substitutes methionine for based chemotherapy as first-line treatment [5, 6, 19, 20], threonine at amino acid position 790 at exon 20 of EGFR, acquired resistance to EGFR TKIs eventually emerges. In was the most commonly acquired resistance mechanism. It 2010, Jackman et al. proposed clinical criteria for acquired accounted for about 50–60% of cases with acquired resist- resistance to EGFR TKI based on the Response Evaluation ance to gefitinib or erlotinib [9, 10]. The790residueisina Criteria in Solid Tumors (RECIST) [21, 22]. Acquired re- key location at the entrance to a hydrophobic pocket of the sistance is defined as when EGFR-mutant NSCLC patients ATP-binding cleft, so it is also referred to as a “gatekeeper” achieved a response or stable disease with greater than six mutation. Because of the bulky methionine sidechain, months of targeted therapy and subsequently developed T790M causes conformational change that leads to the de- disease progression while still on the targeted agent [22]. velopment of steric hindrance and affects the ability of EGFR However, the patterns of disease progression varied in TKItobind tothe ATP-kinase pocket [9]. In addition, the clinical practice. T790M mutation of EGFR could restore the affinity of the Oncologists traditionally change treatment regimens when mutant receptor for ATP, thus reducing the potency of com- there is objective evidence of radiological or clinical progres- petitive inhibitors [27]. sion. However, in routine practice, different characteristics of Other second-point mutations, such as D761Y [28], disease progression might develop when using EGFR TKIs, T854A [29], or L747S [30], confer acquired EGFR TKI resist- and will confuse clinicians. Gandara et al. divided disease pro- ance, although the definite mechanism is still unclear. gression with EGFR TKIs use into three subtypes, including: oligoprogression (new sites or regrowth in a limited number Alternative pathway activation of areas, maximum of four progression sites), systemic pro- Alternative or bypass pathway activation also causes pri- gression (multisite progression), and central nervous system mary resistance. Through bypass tract activation, cancer (CNS) sanctuary progression (excluding leptomeningeal car- cells can survive and proliferate, even when inhibits by the cinomatosis due to the lack of effective treatment options for initial driver pathway. The most common bypass pathway long-term control) [23]. For patients with CNS sanctuary is MET amplification, which accounts for 5–10% of cases progression and/or oligoprogressive disease when using a with acquired resistance to EGFR TKIs [31, 32]. MET gene Wu and Shih Molecular Cancer (2018) 17:38 Page 3 of 14 Fig. 1 The distribution of different acquired resistance mechanisms amplification could activate PI3K-AKT pathway signaling of 37 patients [35]. In terms of morphology, the cancer cells independent of EGFR through driving ERBB3 lost their epithelial features (e.g., E-cadherin expression) dimerization and signaling [31]. However, the threshold of and transformed into spindle-like mesenchymal cells with a MET amplification that would induce TKI resistance has gain of vimentin [45]. not been clarified. Overexpression of hepatocyte growth factor, the ligand of MET oncoprotein, also promotes Exploring the resistance mechanism of EGFR TKIs EGFR TKI resistance [33]. Different mechanisms can be detected in disease progres- Activation of other alternative pathways, including sion to EGFR TKIs [46]. It is important to identify the HER2 amplification [34], PIK3CA mutation [35], BRAF definite tumor resistance mechanism. Repeated tumor bi- mutation, and increased expression of the receptor tyro- opsy is a key factor for the subsequent treatment plan. sine kinase AXL, have been reported to promote ac- Genotyping, whether for the existence of EGFR T790M quired resistance to EGFR TKIs [36]. mutations or other oncogenic alterations, is a crucial step in guiding future treatment, according to the current Histological and phenotypic transformation NSCLC guidelines [47, 48]. About 5% of patients suffered from transformation from However, tumor heterogeneity appears in the primary EGFR-mutant adenocarcinoma to small-cell lung cancer tumor and in metastatic lesions. Intratumor and inter- (SCLC) after acquired resistance to EGFR TKIs [35]. A pos- metastases may have diverse clones with different onco- sible theory is that the initial sample bias resulted in miss- genic driver mutations or resistance mechanisms [49]. The ing the preexisting SCLC component in the original tumor. resistant mutations may occur at a small clone of tumor However, the patient had a good treatment response and cells and clonal evolution may develop during the treat- prolonged PFS [37], and the original activating EGFR muta- ment process, so molecular-based detection methods play tions of adenocarcinoma persisted in the re-biopsy SCLC an important role. Mutation-enriched or ultra-sensitive specimens [38, 39]. Recent studies disclosed that the SCLC (defined as an analytic sensitivity below 1%) molecular- transformation process is predisposed in adenocarcinoma based detection methods should be considered [46, 50]. by inactivation of Rb and p53 [40, 41]. In addition, evalu- The guideline of the College of American Pathologists, ation of the RB1 and TP53 status of adenocarcinoma is pre- International Association for the Study of Lung Cancer, dictive biomarker for SCLC transformation after TKI and Association for Molecular Pathology recommends that treatment [40, 41]. SCLC transformation arises from com- the assay for the EGFR T790M resistant mutation is able to mon progenitor cells of adenocarcinoma in response to detect the mutation in as few as 5% of cells or less (assum- EGFR TKI therapy [37]. ing heterozygosity, a 2.5% mutant allele fraction) in clinical Inappropriate induction of epithelial–mesenchymal tran- practice [50]. For traditional PCR-based methods, Sanger sition (EMT) in tumor cells caused tumor invasion, metas- sequencing provided a sensitivity of only about 20%. Other tasis, drug resistance, and stem cell properties [42, 43]. highly sensitive PCR-based assays utilizing locked nucleic Many studies have shown that EMT is a mechanism of ac- acids (LNAs) or peptide nucleic acids (PNAs) could reach quired resistance to EGFR TKIs. Different EMT transcrip- 0.1–2% of analytical sensitivity [51]. Kinase fusions recently tion factors, including Slug, ZEB1, Snail, and AXL, changed were reported as mechanisms of acquired resistance to with the development of acquired resistance to EGFR TKIs EGFR TKIs [52]. Next-generation sequencing (NGS) is be- [42, 44]. EMT was reported in two (5%) re-biopsy tumors coming the preferred method because it can provide high Wu and Shih Molecular Cancer (2018) 17:38 Page 4 of 14 sensitivity to detect known and unknown mutations and ctDNA testing (Table 1)[59]. The EGFR T790M mutation genetic alterations. could be detected in plasma samples by highly sensitive Sometimes, it is difficult to obtain the re-biopsy tumor genotyping methods, including next-generation sequencing, specimens because of the potential risks of invasive diag- droplet digital polymerase chain reaction (ddPCR), and bead, nostic procedures. Prospective studies showed that the suc- emulsion, amplification and magnetics (BEAMing) assays cess rate of repeated biopsy was 75–95%, and serious [60–63]. TheFDA hasapprovedtheRochereal-timePCR complications were detected in about 1% of cases [32, 53, assay, cobas® EGFR Mutation Test v2, for detection of EGFR 54]. Although repeated biopsy seems safe in clinical prac- mutations in ctDNA in blood samples. Using ctDNA to tice, it is still limited in use because of patient fear and detect mutations can produce a high positive predictive physician preference. Therefore, obtaining serial biopsies value. But, not all tumors shed ctDNA to the same degree, from the same patient is rarely feasible during the NSCLC because of differences in tumor size, stage, location, vascular- treatment course. In addition, the existence of intra-tumor ity, sites of metastatic disease and treatment history [64, 65]. heterogeneity influences tumor evolution, metastasis and Several studies found that up to 35% of patients with EGFR resistance mechanisms in different ways, including somatic T790M might have false-negative plasma levels, compared mutations, epigenetic change and post-transcriptional with tissuebiopsy[66, 67]. Therefore, if liquid biopsy shows modification [55–57]. Therefore, there may be selection anegative EGFR T790M mutation, tissue biopsy for bias because a single snapshot biopsy specimen is not confirmation is necessary [66]. enough to accurately represent all the resistance mecha- Serial analysis of ctDNA can track the molecular dynamic nisms of different sites. evolution of the tumor and monitor treatment response. Liquid biopsy, on the other hand, could provide a source However, the technological approach is not standardized of information on the resistance mutations of the entire because of the broad range of ctDNA isolation techniques, tumor landscape, compared with the single site sampled DNA analysis and quantification [65, 68]. using conventional tumor tissue biopsy [58]. Cell-free circu- lating DNA (ctDNA) is adopted for noninvasive exploration The management of progression during EGFR TKIs use of resistance mechanisms and tumor genetic alterations. According to the NCCN guideline [48], subsequent ther- ctDNA theoretically could provide a surrogate of the whole apy after progression with first-line EGFR TKIs includes tumor genome of both primary and metastatic lesions. different treatment recommendations, which have been Different methodologies, with high sensitivity and detection plotted as an algorithm. For patients with sensitizing of genetic number and type alteration, are being used for EGFR mutations who progress during or after first-line Table 1 Sensitivity of detection of circulating tumor DNA (ctDNA) Test Detection Analytic EGFR T790M mutation Test Characteristics Reference limitation Sensitivity Specificity MS Known SNVs 1–10% 38.9% for del19/L858R 84.6% for del19/L858R Quantitative [122] dHLPC Known SNVs 1–5% 81.8% for sensitizing 89.5% for sensitizing Provided information only of [123, 124] mutation mutation presence/absence of known mutations Cobas Known SNVs 1–3% 61.4% (76.7% for 78.6% Semi-quantitative [70, 71] del19/L858R) (98.2% del19/L858R) The only FDA approved ctDNA assay for detection of EGFR mutations Scorpion-ARMS Known SNVs 1–3% 61.8%–85.7% for 94.3–100% for Semi-quantitative [72, 125] del19/L858R del19/L858R HRMA Known SNVs, 0.1–10% 91.67% for sensitizing 100% for sensitizing Rapid EGFR mutation screening [126] indels, mutation mutation ddPCR Known SNVs > 0.1% 77% (74–82% for 63% (100% for Quantitative Rapid turnaround time [73] del19/L858R) del19/L858R) BEAMing Known SNVs, > 0.1–0.01% 70% 69% Quantitative Detects complex [66] CNVs, SV alteration NGS Known/new SNVs, 0.01%–5% 93% (87–100% 94% (96–100% for Quantitative Profiles large gene panels [127–129] indels, CNVs, SV for del19/L858R) del19/L858R) Detects more complex alteration PNA-PCR Known SNVs, 0.01% 78% for del19/L858R 100% for del19/L858R Semi-quantitative [130, 131] indel, SNV single nucleotide variant, ctDNA circulating tumor DNA, ARMS amplification refractory mutation system, BEAMing beads, emulsion, amplification and magnetics, ddPCR digital droplet polymerase chain reaction, del deletion, indel insertion/deletion, FDA US Food and Drug Administration, NGS next-generation sequencing, CNVs copy number variants, SV structure variants, HRMA high-resolution melting analysis, dHLPC denaturing high performance liquid chromatography, MS mass spectrophotometry (MS), PNA-PCR peptide nucleic acid-polymerase chain reaction Wu and Shih Molecular Cancer (2018) 17:38 Page 5 of 14 targeted therapy, recommended therapy depends on the erlotinib therapy following progression is feasible in se- acquired resistance mechanism and whether the pro- lected patients [75]. The NCCN Panel recommended con- gression is asymptomatic or symptomatic. tinuing EGFR TKIs, whether erlotinib, gefitinib, or afatinib, We modified the latest NCCN and ESMO Guidelines and considering local therapy in patients with asymptom- [48, 69], and included the feasibility of liquid biopsy based atic progression [48]. on the emerging evidence from studies and trials [70–73]. A flare-up phenomenon (rapid disease progression) occa- An algorithm was proposed (Fig. 2) to provide clinical sionally is noted after discontinuation of EGFR TKIs. Intra- physicians with an appropriate practice plan for patients tumor heterogeneity is the possible mechanism of the who experience disease progression on EGFR TKIs. phenomenon. Compared to the resistant clone with indo- lent behavior, rapid regrowth of TKI-sensitive clones causes TKI beyond progression rapid clinical deterioration when EGFR TKIs are discontin- In clinical practice, clinicians may prescribe EGFR TKI ued [76]. One retrospective study also showed that 14 of 61 therapy beyond progression, especially when patients suffer (23%) patients suffered from disease flare after stopping from asymptomatic progression. Nishie et al. retrospectively EGFR TKIs [77]. Therefore, some patients were prescribed analyzed Japanese patients with EGFR mutations. Continu- EGFR TKIs after acquired resistance to EGFR TKIs. ous use of EGFR TKIs beyond progression in patients with The phase III IMPRESS trial aimed to evaluate the effi- activating EGFR mutations may prolong OS compared with cacy and safety of continuing gefitinib combined with switching to cytotoxic chemotherapy [74]. In addition, the chemotherapy versus chemotherapy alone in patients with phase II ASPIRATION study demonstrated that continued EGFR-mutation-positive advanced NSCLC with acquired Fig. 2 Treatment algorism for advanced EGFR-mutant NSCLC patients Wu and Shih Molecular Cancer (2018) 17:38 Page 6 of 14 resistance to first-line gefitinib. A total of 265 patients less epithelial toxicity due to less wild-type EGFR activity [86, were enrolled. However, continuation of gefitinib after dis- 87]. Among them, osimertinib (AZD9291) received FDA ease progression on first-line gefitinib did not prolong PFS and European Medicines Agency (EMA) approval in No- in patients treated with platinum-based doublet chemo- vember 2015 and February 2016, respectively, for treatment therapy as subsequent treatment. A long-term follow-up of patients with T790M mutation-positive NSCLC after ac- found that median OS was 13.4 months in the combin- quired resistance to first-line EGFR TKIs treatment. Table 3 ation arm and 19.5 months in the control arm (HR 1.44; shows the available efficacy data of different third-generation p =0.016) [78]. Besides, the gefitinib group had more side EGFR TKIs in clinical trials. effects and grade 3 or worse AEs. According to the results of the IMPRESS trial, continuation of chemotherapy with  Osimertinib (AZD9291) first-generation EGFR TKIs after acquired resistance to EGFR TKIs is not considered as standard treatment. Osimertinib (AstraZeneca, Macclesfield, UK) is an irre- versible mono-anilino-pyrimidine EGFR TKI that covalently Switch therapy binds to the ATP-binding site, CYS797, of the EGFR tyrosine Repeated biopsy could provide information about the mech- kinase domain. In EGFR recombinant enzyme assays, osi- anism of acquired resistance. If there is no targetable onco- mertinib showed potent activity against diverse activating genic driver mutations/bypass pathways and corresponding EGFR mutations with/without T790M. According to the target medications, chemotherapy is still the standard subse- preclinical data, osimertinib has 200 times greater potency quent treatment after acquired resistance to EGFR TKIs. against L858R/T790M than wild-type EGFR [88]. Two circu- The NCCN guideline offers a treatment algorithm for pa- lating metabolites of osimertinib, AZ5104 and AZ7550, were tients whose disease has progressed on first-line EGFR TKIs. detected, and both had comparable potency to sensitizing Platinum doublet with or without bevacizumab chemother- EGFR mutation and T790M [89]. There was no significant apy should be considered and recommended as second-line difference in pharmacokinetic exposure between Asian and treatment for patients when they suffer from systemic pro- non-Asian patients, showing a minimal food effect [90]. In gression due to acquired resistance to EGFR TKIs. addition, unlike first- and second-generation EGFR TKIs, Two retrospective studies found that for EGFR-mutant pa- osimertinib exposure was not affected by concurrent admin- tients who received platinum-based chemotherapy after istration of omeprazole [91]. disease progression with first-line EGFR TKI treatment, the AURA (NCT01802632) is a phase I/II dose-escalation clin- response rates were 14–18%. Their median PFS with ical trial of osimertinib, which enrolled 253 Asian and west- second-line chemotherapy was about four months [79, 80]. ern NSCLC patients with acquired resistance to first- or Because EGFR mutations are detected mostly in patients second-generation EGFR TKIs, as defined by Jackman cri- with an adenocarcinoma or non-squamous histology, the teria [22, 92]. Patients were not preselected according to optimum regimen might be pemetrexed and platinum T790M status [92]. Thirty-one patients were treated across combination treatment [81], followed by maintenance five dose-escalation cohorts (20, 40, 80, 160 and 240 mg oral, pemetrexed for patients who did not suffer from disease daily) and 222 were treated in the dose-expansion cohort. progression [48, 82]. In the dose-escalation cohort, there was no dose-limiting The most common mechanism of acquired resistance toxicity (DLT) and the maximum tolerated dose (MTD) has to EGFR TKIs is acquired T790M mutation. Second- not been reached. Of the 239 evaluable patients, the object- generation EGFR TKIs, including afatinib, dacomitinib ive response rate (ORR) was 51% and the disease control rate and neratinib, had efficacy in inhibiting proliferation of (DCR) was 84%. Patients with EGFR-T790M mutation had a T790M mutation-positive cells in vitro. However, clinical better ORR (61% vs. 21%), DCR (95% vs. 61%), and longer trials showed disappointing results due to high toxicities median PFS (9.6 months vs. 2.8 months) than patients with- resulting from the narrow therapeutic window. In con- out an EGFR-T790M mutation. The drug is relatively safe, trast to second-generation EGFR TKIs, third-generation and most of the AEs were grade 1 and 2. The most common EGFR TKIs had a good treatment effect on tumors har- AEs were diarrhea (47%), skin toxicity (40%), nausea (22%), boring EGFR T790M mutations [48, 83–85]. and anorexia (21%). When patients took higher dose levels (160 and 240 mg), there was an increasing incidence and se- Next-generation (third-generation) epidermal growth factor verity of AEs (rash, dry skin, and diarrhea). Based on efficacy receptor tyrosine kinases inhibitors (EGFR TKIs) and safety, 80 mg daily was selected as the recommended The third-generation EGFR TKIs can form an irreversible dose for further clinical trials [92]. covalent binding to EGFR. They are pyrimidine-based com- Then, a phase II “AURA2” study (NCT02094261) was pounds, and differ from quinazolines-based first-and second- initiated to enroll NSCLC patients with an EGFR-T790M generation EGFR TKIs (Table 2)[86]. Third-generation mutation and acquired resistance to approved EGFR TKIs; EGFR TKIs can attenuate EGFR T790M activity and have the enrollment criteria were similar to those of the AURA Wu and Shih Molecular Cancer (2018) 17:38 Page 7 of 14 Table 2 Different generations of EGFR TKIs Generation EGFR inhibition Drug Molecular Targets Adverse effect Status 1st-generation Reversible; Gefitinib EGFR del19, L858R Skin rash/acne, abnormal LFT FDA approved competitive Erlotinib EGFR del19, L858R FDA approved 2nd-generation Irreversible; covalent Afatinib EGFR del19, L858R, uncommon Diarrhea, paronychia. FDA approved mutations, HER2, HER4 Skin rash Dacomitinib EGFR del19, L858R, HER2, HER4 Diarrhea, skin rash/acne Phase III Neratinib EGFR G719X, HER2, HER4 Diarrhea, dyspnea, N/V Phase II 3rd-generation Irreversible; Osimertinib EGFR mutations and T790M Diarrhea, skin rash FDA approved covalent Rociletinib EGFR T790M mutation, IGF-1R Hyperglycemia, QTc prolong Withdrawn Olmutinib EGFR T790M mutation Diarrhea, skin exfoliation, Approved in South Korea nausea ASP8273 EGFR L858R, del19, T790M, Diarrhea, N/V, Phase III Discontinued thrombocytopenia Nazartinib EGFR L858R, del19, T790M, Rash, diarrhea, pruritus Phase I/II Avitinib (AC0010) EGFR L858R, del19, T790M, Diarrhea, skin rash, Phase I/II abnormal LFT HS-10296 EGFR sensitive mutations None reported Phase I/II (G719X, del19, L858R, L861Q) +/− T790M PF-06747775 EGFR L858R, del19, T790M, None reported Phase I/II N/V nausea and/or vomiting, LFT liver function test, del19 deletion in exon19, EGFR epidermal growth factor receptor, FDA Food and Drug Administration The targets included FDA approved or associated targets study extension cohort. A preplanned pooled analysis was therapy. The investigator-assessed PFS (primary endpoint) performed, including 201 patients from the 80 mg osimer- was significantly longer in the osimertinib arm than in the tinib expansion cohort of AURA and 210 patients from chemotherapy arm (median 10.1 vs. 4.4 months; HR 0.30; AURA2; ORR was 66%, DCR was 91%, and median PFS p < 0.001). The FDA has granted regular approval to the was 11.0 months [93]. third-generation EGFR TKI, osimertinib, for the treatment In the phase III AURA3 study, 419 patients were ran- of patients with metastatic EGFR T790M mutation-positive domized into osimertinib or platinum-pemetrexed chemo- NSCLC. therapy (maintenance pemetrexed was allowed) groups In the preclinical study, osimertinib demonstrated after they had acquired resistance to first-line EGFR TKI greater penetration of the mouse blood-brain barrier than Table 3 Efficacy of third-generation EGFR TKIs in EGFR T790M-positive NSCLC patients Drug Trial Patients (N) Dose ORR T790M PFS (mo.) Osimertinib AURA phase I [92] Total: 253 T790 20-240 mg QD T790M(+): T790M(+): M(+): 138 61% T790M(−): 21% 9.6 T790M(−): 2.8 AURA phase I T790M(+) 63 80 mg QD 71% 9.7 AURA phase II 210 80 mg QD 70% 9.9 AURA phase II extension [132] 411 80 mg QD 62% 12.3 AURA phase III [84] 416 -Osimertinib 71% 10.1 arm: 279 31% 4.4 -Chemotherapy Odds ratio:5.39 HR: 0.30 arm: 140 (95% CI: 3.47–8.48) (95% CI: 0.23–0.41) Rociletinib TIGER-X phase I/II [98] Total: 69 500, 625 or 45% T790M(+): 9.6 T790M(+): 51 750 mg bid T790M(−): 2.8 Olmutinib HM-EMSI-101 phase 76 800 mg QD 62% 6.9 I/II T790M(+) [133] ASP8273 NCT02113813 phase I/II [134] Total: 63 300 mg QD 29% 6.8 T790M(+): 58 Nazartinib NCT02108964 phase I/II [105] 152 75-350 mg QD 46.9% 9.7 Avitinib (AC0010) NCT02330367 phase I/II [106] 136 50-350 mg QD 44% Wu and Shih Molecular Cancer (2018) 17:38 Page 8 of 14 gefitinib, rociletinib, or afatinib [94]. There were several resistance to osimertinib and rociletinib [103]. A multi- reports of dramatic intracranial response to osimertinib in cohort, phase 1 study (NCT02113813) was initiated to evalu- patients with EGFR T790M lung cancer [94, 95]. A phase ate the safety and efficacy of ASP8273 in NSCLC patients I study (BLOOM, NCT02228369), which has enrolled pre- with disease progression after EGFR TKI treatment. The treated EGFR-mutant NSCLC patients with leptomenin- most common AEs included diarrhea (47%), nausea (42%), geal metastasis treated with 160 mg osimertinib once and fatigue (32%). The most common grade 3/4 AE was daily, is ongoing. The preliminary data is promising [96]. hyponatremia (17%). Across all doses, the ORR was 30.7%, and median PFS was 6.8 months in patients with EGFR Rociletinib (CO-1686) T790M [104]. A phase III randomized clinical trial (SOLAR) was conducted to compare the efficacy and safety of Rociletinib, a 2,4-disubstituted pyrimidine compound, is ASP8273 with that of erlotinib or gefitinib as first-line treat- an oral, irreversible, mutant-selective inhibitor of activating ment for advanced EGFR-mutant NSCLC (NCT02588261). EGFR mutations, including T790M, and spares wild-type However, Astellas Pharma (OTCPK: ALPMY) terminated EGFR [97]. TIGER-X (NCT01526928A), a phase I/II trial of the phase III SOLAR study in May 2017 because the rociletinib, enrolled 130 EGFR-mutant NSCLC patients with treatment advantage apparently was not adequate enough to acquired resistance to first- or second-generation EGFR TKIs justify continuation. [83]. The ORR was 59% for the 46 evaluable T790M mutation-positive patients and 29% for the 17 T790M  Nazartinib (EGF816) mutation-negative patients [83]. Because of targeting of IGF- 1R, hyperglycemia (22%) was detected as the most common A phase I/II first-in-human study, NCT02108964 grade 3 AE. An independent updated analysis of the TIGER- (EGF816X2101), investigated nazartinib in EGFR-mutant X trial showed that the T790M mutation-positive patients patients. A total of 152 patients were treated across seven had an ORR of 45% [98]. In addition, a series of cases with cohorts using doses ranging from 75 to 350 mg [105]. response to osimertinib after resistance to rociletinib were Among the 147 evaluable patients, the ORR and DCR reported [99]. Clovis Oncology, Inc. decided to stop enroll- were 46.9% and 87.1%, respectively. The median PFS ment in all ongoing rociletinib studies and terminate the fu- across all dose cohorts was 9.7 months. Skin rash (54%), ture development program in May 2016. diarrhea (37%), and pruritus (34%) were the most com- mon AEs. The skin rashes related to nazartinib were dif- Olmutinib (BI-1482694/HM61713; Olita™) ferent from those caused by other EGFR TKIs in pattern, location, and histology. The most common grade 3/4 AE A phase I/II dose escalation clinical trial, HM-EMSI-101 was diarrhea (16%) [105]. A phase II clinical trial with six (NCT01588145), was initiated in South Korea [100]. Pa- cohorts is ongoing. In addition, a phase Ib/II trial tients took olmutinib in doses ranging from 75 to 1200 mg/ (NCT02335944 and NCT02323126) is ongoing to investi- day. Among the 34 patients with NSCLC harboring T790M gate the efficacy of combined treatments with INC280, a detected by a central laboratory, the ORR was 58.8%. The specific MET inhibitor, and with nivolumab, an anti-PD-1 DCR was 97.1% for patients treated with olmutinib in doses monoclonal antibody in patients with EGFR-T790M mu- greater than 650 mg. The most common DLTs involved tation after acquired resistance to first-line EGFR TKI. gastrointestinal symptoms, abnormal liver function (AST/ ALT), and increasing amylase/lipase levels. Therefore,  AC0010 800 mg/day was selected as the recommended phase II dose. Seventy-six patients with centrally confirmed T790M A phase I/II, first-in-human dose-escalation and expan- mutation-positive NSCLC were enrolled in part II of the sion phase clinical trial (NCT02330367) was carried out study, and 70 were evaluable for response. The ORR was with advanced NSCLC patients with acquired T790M mu- 61% and median PFS was 6.9 months [101]. Based on the tation after first-generation EGFR TKIs treatment [106]. In aforementioned result, olmutinib was first approved in all, 136 patients have been treated across seven cohorts (50, South Korea in 2016. However, Boehringer Ingelheim 100, 150, 200, 250, 300, and 350 mg BID), and MTD has decided to stop the co-development of this drug because of not been reached. The most common drug-related AEs an unexpected grade 3/4 skin toxicity (including palmo- were diarrhea (38%), rash (26%) and ALT/AST elevation. plantar keratoderma) [102]. Grade3/4 AEsof diarrhea(2%), rash (2%) andALT/AST elevation (4%, 2%) were recorded. The 124 evaluable ASP8273 patients had ORR and DCR of 44% and 85%, respectively. Because of the drug safety profile and activity against Preclinical data showed ASP8273 had antitumor activity NSCLC with acquired T790M mutation, a phase II, against EGFR TKI-resistant cells, including those with AEGIS-1 study is ongoing to evaluate treatment efficacy for Wu and Shih Molecular Cancer (2018) 17:38 Page 9 of 14 patients with T790M mutation-positive NSCLC with T790M-positive and T790M-negative tumors was compar- acquired resistance to first-generation EGFR TKIs. An open able (32% vs. 25%; p = .341). The two groups showed no stat- label, randomized phase III trial (NCT03058094) also is on- istical difference in PFS. The NCCN Panel recommends going to compare AC0010 (300 mg, BID) with pemetrexed/ considering an afatinib/cetuximab regimen for patients who cisplatin (4–6 cycles) in patients with advanced NSCLC have progressed after receiving EGFR TKIs and chemother- who have progressed following prior therapy with EGFR apy [48]. However, skin rash (90% all grades) and diarrhea TKI. T790M in biopsy samples was confirmed by a central (71% all grades) were the two most common adverse effects. laboratory. Grades 3 and 4 adverse effects were 44% and 2%, respect- ively. Because of the high rate of AEs with this combination HS-10296 therapy, it is no longer a preferred treatment for patients with tumor harboring EGFR T790M mutations [110]. An open-label, multicenter, phase I/II dose escalation and expansion trial (NCT02981108) is currently recruiting patients  Horizontal pathway with locally advanced or metastatic NSCLC after acquired resistance to first- and/or second-generation EGFR TKIs. Since bypass signaling pathway activation is an important acquired resistance mechanism of EGFR TKIs, it is reason- PF-06747775 able to combine inhibition of EGFR pathway signaling and inhibitors for the bypass signaling pathway to overcome re- PF-06747775 has potent antitumor efficacy against sistance. Different horizontal combination strategies are be- NSCLC harboring a classical mutation with/without ing investigated, but results are preliminary and immature T790M. It significantly attenuates T790M activity and has (Table 4). less toxicity because of the reduction of proteome reactivity MET amplification is an important mechanism of acquired relative to earlier EGFR TKIs [107, 108]. A phase I/II clin- resistance to EGFR TKI therapy [31, 111]. A randomized, ical trial (NCT02349633) involving patients with advanced open-label, phase 2 study enrolled patients with advanced NSCLC harboring EGFR mutations (Del19 or L858R with/ NSCLC (enriched for EGFR-mutant disease) who developed without T790M) is ongoing. acquired resistance to erlotinib to receive emibetuzumab (LY2875358), a humanized IgG4 monoclonal bivalent MET Combination therapy antibody, with or without erlotinib therapy. The ORR of pa- tients whose re-biopsy samples harbored MET overexpres- Vertical pathway sion (≥60%) was 3.8% in the combination arm and 4.8% in the monotherapy arm [112]. In Japan, another phase II Cetuximab is a recombinant human/mouse chimeric clinical trial enrolled 45 patients with advanced EGFR-mu- EGFR IgG1 monoclonal antibody. Combining afatinib and tant NSCLC who developed acquired resistance to first- cetuximab may be useful for patients who have progressed generation EGFR TKIs to receive tivantinib (ARQ197) and after receiving EGFR TKI therapy and chemotherapy [109]. erlotinib combination therapy. The response rate was Among 126 patients, the response rate of patients with 6.7%. High MET expression (≥ 50%) was detected by Table 4 Main mechanisms involved in acquired resistance to EGF receptor-tyrosine kinase inhibitors and the associated targetable drugs Molecular alteration Pathway Targetable drug HER2 amplification Afatinib, Trastuzumab, ado-trastuzumab emtansine (TDM1) MET overexpression/genetic alteration ● Anti-HGF antibody: Rilotumumab, Ficlatuzumab ● Anti-c-MET antibody: MET Mab, Emibetuzumab (LY2875358) ● Selective c-MET inhibitor: Tivantinib (ARQ197), Capmatinib (INC280), Savolitinib (AZD6094), Tepotinib (EMD 1214063), SGX523, SAR125844, ● Multikinase inhibitors: Crizotinib, Cabozantinib (XL184), Glesatinib (MGCD265), Merestinib (LY2801653), S49076 PIK3CA PI3K-AKT-mTOR ● PI3K inhibitor: Pilaralisib (XL147), Dactolisib (BEZ235) and Pictilisib (GDC-0941), Buparlisib (BKM120) ● AKT inhibitor: MK-2206 ● mTOR inhibitor: Everolimus, Temsirolimus, Ridaforolimus BRAF Ras-Raf-MEK-ERK Vemurafenib (PLX4032), Dabrafenib (GSK2118436), Selumetinib, LY3009120 AXL overexpression GAS6-AXL ● Tyrosine kinase inhibitor: Cabozantinib (XL 184) ● AXL antibody: E8, D9, Mab173 ● AXL decoy receptor: AXL-Fc, MYDI Wu and Shih Molecular Cancer (2018) 17:38 Page 10 of 14 immunohistochemical stain in 48.9% of the patients, includ- suppress T-cell function [120]. This finding indicates that ing all three partial responders [113]. In addition, a combin- EGFR functions as an oncogene through cell-autonomous ation of capmatinib (INC280) and gefitinib was tested in a mechanisms and raises the possibility that other oncogenes phase 2 study (NCT01610336) in EGFR-mutant NSCLC may drive immune escape [120]. However, retrospective patients after acquired resistance to gefitinib. EGFR T790M studies showed that NSCLCs harboring EGFR mutations NSCLCs were excluded and high cMET expression was re- were associated with low response rates to PD-1/PD-L1 in- quired. Of the 65 evaluable patients, the ORR was 18% and hibitors, which may have resulted from low rates of concur- DCR was 80%. More responses were seen in tumors with rent PD-L1 expression and CD8(+) TILs within the tumor MET amplifications [114]. microenvironment [119]. A retrospective study on the In addition to MET amplification, different medications are efficacy of nivolumab in patients with EGFR mutation- being investigated to inhibit other bypass signaling pathways, positive NSCLC after EGFR TKI failure found that T790M- including a heat shock protein 90 inhibitor, AUY922 (- negative patients were more likely than T790M-positive ClinicalTrials.gov: NCT01259089 and NCT01646125); a patients to benefit from nivolumab [121]. JAK inhibitor, ruxolitinib (ClinicalTrials.gov: NCT02155465 Different phase 1 trials combining EGFR TKIs with and NCT02145637); a MET/AXL/FGFR inhibitor S- 49076 immunotherapies include nivolumab (ClinicalTrials.gov, (EU Clinical Trials Register:EudraCT Number:2015– number NCT01454102); pembrolizumab (ClinicalTrials.gov, 002646-31) and a PI3K inhibitor, buparlisib (BKM120) number NCT02039674); and atezolizumab (ClinicalTrials.- (ClinicalTrials.gov: NCT01570296 and NCT01487265). gov, number NCT02013219). These studies are all ongoing. Furthermore, combination therapy with osimertinib has been investigated. The TATTON study (ClinicalTrials.gov: Conclusions NCT02143466) enrolled patients who received osimertinib- EGFR TKIs are currently the standard first-line treatment based combination therapy with either a MET inhibitor of patients with advanced NSCLC harboring activating (savolitinib), MEK inhibitor (selumetinib), or anti-PD-L1 EGFR mutations. After acquiring resistance to first-line monoclonal antibody (durvalumab) [115]. However, the EGFR TKI therapy, it is important that the mechanisms of rate of drug-related interstitial disease was high in the osi- acquired resistance in all patients are explored. Then, mertinib plus durvalumab arm, so the development of this based on the mechanism, subsequent treatment can be combination therapy was discontinued [116]. Other clinical chosen. Continuation of EGFR TKI therapy is suitable for trials, including osimertinib in combination with select patients with asymptomatic progression and/or oli- ramucirumab, necitumumab, bevacizumab, or navitoclax goprogression. Repeat tumor biopsy to detect the EGFR (ClinicalTrials.gov, NCT02789345, 02496663, 02803203 T790M mutation is the current standard of care, and osi- and 02520778), are ongoing. mertinib has been approved for patients with acquired Combination therapies have higher rates of toxicities EGFR T790M-mutant disease. Liquid biopsy is an alterna- and side effects than a single agent does. Although the tive method to detect plasma EGFR T790M mutation and aforementioned medications have been evaluated in to identify patients suitable for osimertinib therapy. Com- clinical trials, clinicians should keep in mind the bination therapy may be effective for acquired resistance possibility of AEs when prescribing combination therapy. resulting from activation of the bypass signaling pathway. Advances in the detection method for different resistance Immunotherapy mechanisms and the development of new drugs are both For subsequent therapy, or immunotherapy, nivolumab and urgently needed for personalized therapy. pembrolizumab have been approved as standard treatment, Abbreviations and high-level PD-L1 expression in tumors can predict a 95% CI: 95% confidence interval; AEs: adverse effects; ASCO: American Society of higher response rate. Phase III trials assessing pembrolizu- Clinical Oncology; ctDNA: circulating tumor DNA; DLT: dose-limiting toxicity; mab, nivolumab, or atezolizumab compared to docetaxel as EGFR: epidermal growth factor receptor; EMT: epithelial–mesenchymal transition; ESMO: European Society for Medical Oncology; FDA: Food and Drug Administration; subsequent therapy for patients with metastatic NSCLC MLT: maximum tolerated dose; NCCN: National Comprehensive Cancer Network; found there were no survival benefits for EGFR-mutant NSCLC: non-small cell lung cancer; ORR: objective response rate, DCR disease control lung cancer patients. Also, there were not enough patients rate; OS: overall survival; PD: progressive disease; PFS: progression-free survival; SCLC: small cell lung cancer; TKI: tyrosine kinase inhibitor with these mutations to determine whether there were sta- tistically significant differences. However, immunotherapy Funding was comparable to chemotherapy and was better tolerated. This research did not receive any specific grant from funding agencies in the [117–119]. Until now, there is not enough evidence to public, commercial, or not-for-profit sectors. recommend pembrolizumab, nivolumab, or atezolizumab Authors’ contributions as subsequent therapy for EGFR-mutant patients. SGW and JYS both completed data collection, literature search, generation of In vitro, EGFR-mutant lung cancer cells inhibited antitu- figures, and writing of the manuscript. Both authors read and approved the mor immunity by activating the PD-1/PD-L1 pathway to final manuscript. Wu and Shih Molecular Cancer (2018) 17:38 Page 11 of 14 Competing interests 16. Piotrowska Z, Thress KS, Mooradian M, Heist RS, Azzoli CG, Temel JS, Rizzo C, Jin-Yuan Shih has received speaking honoraria from AstraZeneca, Roche, Pfizer, Nagy RJ, Lanman RB, Gettinger SN, et al. MET amplification (amp) as a Boehringer Ingelheim, Novartis, and Eli Lilly. 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Management of acquired resistance to EGFR TKI–targeted therapy in advanced non-small cell lung cancer

Molecular Cancer , Volume 17 (1): 14 – Dec 1, 2018

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References (157)

Publisher
Springer Journals
Copyright
2018 The Author(s).
eISSN
1476-4598
DOI
10.1186/s12943-018-0777-1
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Abstract

Recent advances in diagnosis and treatment are enabling a more targeted approach to treating lung cancers. Therapy targeting the specific oncogenic driver mutation could inhibit tumor progression and provide a favorable prognosis in clinical practice. Activating mutations of epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) are a favorable predictive factor for EGFR tyrosine kinase inhibitors (TKIs) treatment. For lung cancer patients with EGFR-exon 19 deletions or an exon 21 Leu858Arg mutation, the standard first-line treatment is first- generation (gefitinib, erlotinib), or second-generation (afatinib) TKIs. EGFR TKIs improve response rates, time to progression, and overall survival. Unfortunately, patients with EGFR mutant lung cancer develop disease progression after a median of 10 to 14 months on EGFR TKI. Different mechanisms of acquired resistance to first-generation and second-generation EGFR TKIs have been reported. Optimal treatment for the various mechanisms of acquired resistance is not yet clearly defined, except for the T790M mutation. Repeated tissue biopsy is important to explore resistance mechanisms, but it has limitations and risks. Liquid biopsy is a valid alternative to tissue re-biopsy. Osimertinib has been approved for patients with T790M-positive NSCLC with acquired resistance to EGFR TKI. For other TKI-resistant mechanisms, combination therapy may be considered. In addition, the use of immunotherapy in lung cancer treatment has evolved rapidly. Understanding and clarifying the biology of the resistance mechanisms of EGFR-mutant NSCLC could guide future drug development, leading to more precise therapy and advances in treatment. Background growth factor receptor (EGFR) mutation-positive patients. In the United States, an additional 224,390 new lung cancer EGFR mutation-positive patients with lung adenocarcin- cases were diagnosed in 2016, and accounted for about 27% oma had aresponserateas highas 80%,and around 10– of all cancer deaths [1]. Although standard platinum-based 14 months of progression-free survival (PFS) [5, 6]. The chemotherapy is the cornerstone of systemic therapy, it has American Society of Clinical Oncology (ASCO), European amodesteffectonoverall survival (OS) [2]. Lung cancer is Society for Medical Oncology (ESMO) and National still the leading cause of cancer death worldwide [3]. Comprehensive Cancer Network (NCCN) guidelines rec- In the most recent decade, treatment of non-small cell ommend EGFR TKIs as first-line treatment for EGFR-mu- lung cancer (NSCLC) has evolved to a great extent. The tant patients. The most common activating mutations are discovery of driver mutations in lung cancer allows the cre- in-frame deletions in exon 19 and single-point mutation of ation of personalized targeted treatment. It is important exon 21 (Leu858Arg), which together account for more that lung cancer patients are tested for oncogenic drivers of than 80% of known activating EGFR mutations [7, 8]. cancer and receive matched targeted therapy [4]. Epidermal Although EGFR TKIs have a favorable and durable treat- growth factor receptor tyrosine kinase inhibitors (EGFR ment response, most patients will eventually develop pro- TKIs) provide a favorable treatment outcome in epidermal gressive disease (PD) within about one year of treatment. Furthermore, acquired resistance develops and limits the long-term efficacy of these EGFR TKIs. A variety of mecha- * Correspondence: jyshih@ntu.edu.tw nisms of acquired resistance to EGFR TKIs have been Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 100, Taiwan © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wu and Shih Molecular Cancer (2018) 17:38 Page 2 of 14 reported. The most common mechanism is the develop- previously beneficial EGFR TKI, it maybereasonabletocon- ment of acquired EGFR T790M mutation [9]. T790M was sider local treatment and continuation of the targeted agent. found in about 50% of EGFR–mutant cases that acquired This approach yielded more than six months of additional resistance to EGFR TKIs [9]. Patients using either first- or disease control [24, 25]. second-generation EGFR TKIs had a similar prevalence of Yang et al. proposed another criteria for EGFR TKI failure acquired T790M [10]. modes in NSCLC [26]. Based on the duration of disease Preclinical data showed that the second-generation control, the evolution of the tumor burden, and clinical EGFR TKI, afatinib, could overcome the resistance caused symptoms, regardless of genotype profile, the diversity of by the T790M mutation [11], but clinical trials have not EGFR TKI failure could be categorized into three modes, revealed the effect due to toxicity limitations. The narrow including dramatic progression, gradual progression, and therapeutic window of afatinib caused severe adverse ef- local progression. The median PFS was 9.3, 12.9, and fects (AEs), probably owing to inhibition of wild-type 9.2 months (p = 0.007) for these three modes, respectively, EGFR [12, 13]. In the phase III LUX-Head & Neck 1 and median OS was 17.7, 39.4, and 23.1 months (p < 0.001), (LHN1) trial, second-line afatinib significantly improved respectively. In patients with disease in the gradual progres- PFS versus methotrexate in patients with recurrent/meta- sion mode, continuing EGFR TKI therapy was superior to static head and neck squamous cell carcinoma [14]. This switching to chemotherapy in terms of OS (39.4 vs. suggests afatinib is a drug active against wild-type EGFR. 17.8 months; p = 0.02) [26]. Determination of the clinical The third-generation EGFR TKI, osimertinib, has been mode could favor strategies for subsequent treatment and approved for patients with T790M-positive NSCLC with prediction of survival. acquired resistance to EGFR TKIs. Use of third-generation EGFR TKIs was related to different acquired resistance Mechanisms of acquired resistance to EGFR TKIs mechanisms [15–18]. Therefore, in this manuscript, we Acquired resistance mechanisms vary. Several study groups focused on these recently developed treatment strategies comprehensively explored the mechanisms through re- for EGFR-mutant NSCLC with acquired resistance to biopsy tissue specimens. The most common acquired first- or second-generation EGFR TKIs. resistance mechanisms were of three types: target gene modification, alternative pathway activation and histological Clinical presentation of acquired resistance to first-line or phenotypic transformation (Fig. 1). EGFR TKIs Although EGFR-mutant patients receiving EGFR TKIs Target gene modification have longer median PFS than those receiving platinum- The T790M mutation, which substitutes methionine for based chemotherapy as first-line treatment [5, 6, 19, 20], threonine at amino acid position 790 at exon 20 of EGFR, acquired resistance to EGFR TKIs eventually emerges. In was the most commonly acquired resistance mechanism. It 2010, Jackman et al. proposed clinical criteria for acquired accounted for about 50–60% of cases with acquired resist- resistance to EGFR TKI based on the Response Evaluation ance to gefitinib or erlotinib [9, 10]. The790residueisina Criteria in Solid Tumors (RECIST) [21, 22]. Acquired re- key location at the entrance to a hydrophobic pocket of the sistance is defined as when EGFR-mutant NSCLC patients ATP-binding cleft, so it is also referred to as a “gatekeeper” achieved a response or stable disease with greater than six mutation. Because of the bulky methionine sidechain, months of targeted therapy and subsequently developed T790M causes conformational change that leads to the de- disease progression while still on the targeted agent [22]. velopment of steric hindrance and affects the ability of EGFR However, the patterns of disease progression varied in TKItobind tothe ATP-kinase pocket [9]. In addition, the clinical practice. T790M mutation of EGFR could restore the affinity of the Oncologists traditionally change treatment regimens when mutant receptor for ATP, thus reducing the potency of com- there is objective evidence of radiological or clinical progres- petitive inhibitors [27]. sion. However, in routine practice, different characteristics of Other second-point mutations, such as D761Y [28], disease progression might develop when using EGFR TKIs, T854A [29], or L747S [30], confer acquired EGFR TKI resist- and will confuse clinicians. Gandara et al. divided disease pro- ance, although the definite mechanism is still unclear. gression with EGFR TKIs use into three subtypes, including: oligoprogression (new sites or regrowth in a limited number Alternative pathway activation of areas, maximum of four progression sites), systemic pro- Alternative or bypass pathway activation also causes pri- gression (multisite progression), and central nervous system mary resistance. Through bypass tract activation, cancer (CNS) sanctuary progression (excluding leptomeningeal car- cells can survive and proliferate, even when inhibits by the cinomatosis due to the lack of effective treatment options for initial driver pathway. The most common bypass pathway long-term control) [23]. For patients with CNS sanctuary is MET amplification, which accounts for 5–10% of cases progression and/or oligoprogressive disease when using a with acquired resistance to EGFR TKIs [31, 32]. MET gene Wu and Shih Molecular Cancer (2018) 17:38 Page 3 of 14 Fig. 1 The distribution of different acquired resistance mechanisms amplification could activate PI3K-AKT pathway signaling of 37 patients [35]. In terms of morphology, the cancer cells independent of EGFR through driving ERBB3 lost their epithelial features (e.g., E-cadherin expression) dimerization and signaling [31]. However, the threshold of and transformed into spindle-like mesenchymal cells with a MET amplification that would induce TKI resistance has gain of vimentin [45]. not been clarified. Overexpression of hepatocyte growth factor, the ligand of MET oncoprotein, also promotes Exploring the resistance mechanism of EGFR TKIs EGFR TKI resistance [33]. Different mechanisms can be detected in disease progres- Activation of other alternative pathways, including sion to EGFR TKIs [46]. It is important to identify the HER2 amplification [34], PIK3CA mutation [35], BRAF definite tumor resistance mechanism. Repeated tumor bi- mutation, and increased expression of the receptor tyro- opsy is a key factor for the subsequent treatment plan. sine kinase AXL, have been reported to promote ac- Genotyping, whether for the existence of EGFR T790M quired resistance to EGFR TKIs [36]. mutations or other oncogenic alterations, is a crucial step in guiding future treatment, according to the current Histological and phenotypic transformation NSCLC guidelines [47, 48]. About 5% of patients suffered from transformation from However, tumor heterogeneity appears in the primary EGFR-mutant adenocarcinoma to small-cell lung cancer tumor and in metastatic lesions. Intratumor and inter- (SCLC) after acquired resistance to EGFR TKIs [35]. A pos- metastases may have diverse clones with different onco- sible theory is that the initial sample bias resulted in miss- genic driver mutations or resistance mechanisms [49]. The ing the preexisting SCLC component in the original tumor. resistant mutations may occur at a small clone of tumor However, the patient had a good treatment response and cells and clonal evolution may develop during the treat- prolonged PFS [37], and the original activating EGFR muta- ment process, so molecular-based detection methods play tions of adenocarcinoma persisted in the re-biopsy SCLC an important role. Mutation-enriched or ultra-sensitive specimens [38, 39]. Recent studies disclosed that the SCLC (defined as an analytic sensitivity below 1%) molecular- transformation process is predisposed in adenocarcinoma based detection methods should be considered [46, 50]. by inactivation of Rb and p53 [40, 41]. In addition, evalu- The guideline of the College of American Pathologists, ation of the RB1 and TP53 status of adenocarcinoma is pre- International Association for the Study of Lung Cancer, dictive biomarker for SCLC transformation after TKI and Association for Molecular Pathology recommends that treatment [40, 41]. SCLC transformation arises from com- the assay for the EGFR T790M resistant mutation is able to mon progenitor cells of adenocarcinoma in response to detect the mutation in as few as 5% of cells or less (assum- EGFR TKI therapy [37]. ing heterozygosity, a 2.5% mutant allele fraction) in clinical Inappropriate induction of epithelial–mesenchymal tran- practice [50]. For traditional PCR-based methods, Sanger sition (EMT) in tumor cells caused tumor invasion, metas- sequencing provided a sensitivity of only about 20%. Other tasis, drug resistance, and stem cell properties [42, 43]. highly sensitive PCR-based assays utilizing locked nucleic Many studies have shown that EMT is a mechanism of ac- acids (LNAs) or peptide nucleic acids (PNAs) could reach quired resistance to EGFR TKIs. Different EMT transcrip- 0.1–2% of analytical sensitivity [51]. Kinase fusions recently tion factors, including Slug, ZEB1, Snail, and AXL, changed were reported as mechanisms of acquired resistance to with the development of acquired resistance to EGFR TKIs EGFR TKIs [52]. Next-generation sequencing (NGS) is be- [42, 44]. EMT was reported in two (5%) re-biopsy tumors coming the preferred method because it can provide high Wu and Shih Molecular Cancer (2018) 17:38 Page 4 of 14 sensitivity to detect known and unknown mutations and ctDNA testing (Table 1)[59]. The EGFR T790M mutation genetic alterations. could be detected in plasma samples by highly sensitive Sometimes, it is difficult to obtain the re-biopsy tumor genotyping methods, including next-generation sequencing, specimens because of the potential risks of invasive diag- droplet digital polymerase chain reaction (ddPCR), and bead, nostic procedures. Prospective studies showed that the suc- emulsion, amplification and magnetics (BEAMing) assays cess rate of repeated biopsy was 75–95%, and serious [60–63]. TheFDA hasapprovedtheRochereal-timePCR complications were detected in about 1% of cases [32, 53, assay, cobas® EGFR Mutation Test v2, for detection of EGFR 54]. Although repeated biopsy seems safe in clinical prac- mutations in ctDNA in blood samples. Using ctDNA to tice, it is still limited in use because of patient fear and detect mutations can produce a high positive predictive physician preference. Therefore, obtaining serial biopsies value. But, not all tumors shed ctDNA to the same degree, from the same patient is rarely feasible during the NSCLC because of differences in tumor size, stage, location, vascular- treatment course. In addition, the existence of intra-tumor ity, sites of metastatic disease and treatment history [64, 65]. heterogeneity influences tumor evolution, metastasis and Several studies found that up to 35% of patients with EGFR resistance mechanisms in different ways, including somatic T790M might have false-negative plasma levels, compared mutations, epigenetic change and post-transcriptional with tissuebiopsy[66, 67]. Therefore, if liquid biopsy shows modification [55–57]. Therefore, there may be selection anegative EGFR T790M mutation, tissue biopsy for bias because a single snapshot biopsy specimen is not confirmation is necessary [66]. enough to accurately represent all the resistance mecha- Serial analysis of ctDNA can track the molecular dynamic nisms of different sites. evolution of the tumor and monitor treatment response. Liquid biopsy, on the other hand, could provide a source However, the technological approach is not standardized of information on the resistance mutations of the entire because of the broad range of ctDNA isolation techniques, tumor landscape, compared with the single site sampled DNA analysis and quantification [65, 68]. using conventional tumor tissue biopsy [58]. Cell-free circu- lating DNA (ctDNA) is adopted for noninvasive exploration The management of progression during EGFR TKIs use of resistance mechanisms and tumor genetic alterations. According to the NCCN guideline [48], subsequent ther- ctDNA theoretically could provide a surrogate of the whole apy after progression with first-line EGFR TKIs includes tumor genome of both primary and metastatic lesions. different treatment recommendations, which have been Different methodologies, with high sensitivity and detection plotted as an algorithm. For patients with sensitizing of genetic number and type alteration, are being used for EGFR mutations who progress during or after first-line Table 1 Sensitivity of detection of circulating tumor DNA (ctDNA) Test Detection Analytic EGFR T790M mutation Test Characteristics Reference limitation Sensitivity Specificity MS Known SNVs 1–10% 38.9% for del19/L858R 84.6% for del19/L858R Quantitative [122] dHLPC Known SNVs 1–5% 81.8% for sensitizing 89.5% for sensitizing Provided information only of [123, 124] mutation mutation presence/absence of known mutations Cobas Known SNVs 1–3% 61.4% (76.7% for 78.6% Semi-quantitative [70, 71] del19/L858R) (98.2% del19/L858R) The only FDA approved ctDNA assay for detection of EGFR mutations Scorpion-ARMS Known SNVs 1–3% 61.8%–85.7% for 94.3–100% for Semi-quantitative [72, 125] del19/L858R del19/L858R HRMA Known SNVs, 0.1–10% 91.67% for sensitizing 100% for sensitizing Rapid EGFR mutation screening [126] indels, mutation mutation ddPCR Known SNVs > 0.1% 77% (74–82% for 63% (100% for Quantitative Rapid turnaround time [73] del19/L858R) del19/L858R) BEAMing Known SNVs, > 0.1–0.01% 70% 69% Quantitative Detects complex [66] CNVs, SV alteration NGS Known/new SNVs, 0.01%–5% 93% (87–100% 94% (96–100% for Quantitative Profiles large gene panels [127–129] indels, CNVs, SV for del19/L858R) del19/L858R) Detects more complex alteration PNA-PCR Known SNVs, 0.01% 78% for del19/L858R 100% for del19/L858R Semi-quantitative [130, 131] indel, SNV single nucleotide variant, ctDNA circulating tumor DNA, ARMS amplification refractory mutation system, BEAMing beads, emulsion, amplification and magnetics, ddPCR digital droplet polymerase chain reaction, del deletion, indel insertion/deletion, FDA US Food and Drug Administration, NGS next-generation sequencing, CNVs copy number variants, SV structure variants, HRMA high-resolution melting analysis, dHLPC denaturing high performance liquid chromatography, MS mass spectrophotometry (MS), PNA-PCR peptide nucleic acid-polymerase chain reaction Wu and Shih Molecular Cancer (2018) 17:38 Page 5 of 14 targeted therapy, recommended therapy depends on the erlotinib therapy following progression is feasible in se- acquired resistance mechanism and whether the pro- lected patients [75]. The NCCN Panel recommended con- gression is asymptomatic or symptomatic. tinuing EGFR TKIs, whether erlotinib, gefitinib, or afatinib, We modified the latest NCCN and ESMO Guidelines and considering local therapy in patients with asymptom- [48, 69], and included the feasibility of liquid biopsy based atic progression [48]. on the emerging evidence from studies and trials [70–73]. A flare-up phenomenon (rapid disease progression) occa- An algorithm was proposed (Fig. 2) to provide clinical sionally is noted after discontinuation of EGFR TKIs. Intra- physicians with an appropriate practice plan for patients tumor heterogeneity is the possible mechanism of the who experience disease progression on EGFR TKIs. phenomenon. Compared to the resistant clone with indo- lent behavior, rapid regrowth of TKI-sensitive clones causes TKI beyond progression rapid clinical deterioration when EGFR TKIs are discontin- In clinical practice, clinicians may prescribe EGFR TKI ued [76]. One retrospective study also showed that 14 of 61 therapy beyond progression, especially when patients suffer (23%) patients suffered from disease flare after stopping from asymptomatic progression. Nishie et al. retrospectively EGFR TKIs [77]. Therefore, some patients were prescribed analyzed Japanese patients with EGFR mutations. Continu- EGFR TKIs after acquired resistance to EGFR TKIs. ous use of EGFR TKIs beyond progression in patients with The phase III IMPRESS trial aimed to evaluate the effi- activating EGFR mutations may prolong OS compared with cacy and safety of continuing gefitinib combined with switching to cytotoxic chemotherapy [74]. In addition, the chemotherapy versus chemotherapy alone in patients with phase II ASPIRATION study demonstrated that continued EGFR-mutation-positive advanced NSCLC with acquired Fig. 2 Treatment algorism for advanced EGFR-mutant NSCLC patients Wu and Shih Molecular Cancer (2018) 17:38 Page 6 of 14 resistance to first-line gefitinib. A total of 265 patients less epithelial toxicity due to less wild-type EGFR activity [86, were enrolled. However, continuation of gefitinib after dis- 87]. Among them, osimertinib (AZD9291) received FDA ease progression on first-line gefitinib did not prolong PFS and European Medicines Agency (EMA) approval in No- in patients treated with platinum-based doublet chemo- vember 2015 and February 2016, respectively, for treatment therapy as subsequent treatment. A long-term follow-up of patients with T790M mutation-positive NSCLC after ac- found that median OS was 13.4 months in the combin- quired resistance to first-line EGFR TKIs treatment. Table 3 ation arm and 19.5 months in the control arm (HR 1.44; shows the available efficacy data of different third-generation p =0.016) [78]. Besides, the gefitinib group had more side EGFR TKIs in clinical trials. effects and grade 3 or worse AEs. According to the results of the IMPRESS trial, continuation of chemotherapy with  Osimertinib (AZD9291) first-generation EGFR TKIs after acquired resistance to EGFR TKIs is not considered as standard treatment. Osimertinib (AstraZeneca, Macclesfield, UK) is an irre- versible mono-anilino-pyrimidine EGFR TKI that covalently Switch therapy binds to the ATP-binding site, CYS797, of the EGFR tyrosine Repeated biopsy could provide information about the mech- kinase domain. In EGFR recombinant enzyme assays, osi- anism of acquired resistance. If there is no targetable onco- mertinib showed potent activity against diverse activating genic driver mutations/bypass pathways and corresponding EGFR mutations with/without T790M. According to the target medications, chemotherapy is still the standard subse- preclinical data, osimertinib has 200 times greater potency quent treatment after acquired resistance to EGFR TKIs. against L858R/T790M than wild-type EGFR [88]. Two circu- The NCCN guideline offers a treatment algorithm for pa- lating metabolites of osimertinib, AZ5104 and AZ7550, were tients whose disease has progressed on first-line EGFR TKIs. detected, and both had comparable potency to sensitizing Platinum doublet with or without bevacizumab chemother- EGFR mutation and T790M [89]. There was no significant apy should be considered and recommended as second-line difference in pharmacokinetic exposure between Asian and treatment for patients when they suffer from systemic pro- non-Asian patients, showing a minimal food effect [90]. In gression due to acquired resistance to EGFR TKIs. addition, unlike first- and second-generation EGFR TKIs, Two retrospective studies found that for EGFR-mutant pa- osimertinib exposure was not affected by concurrent admin- tients who received platinum-based chemotherapy after istration of omeprazole [91]. disease progression with first-line EGFR TKI treatment, the AURA (NCT01802632) is a phase I/II dose-escalation clin- response rates were 14–18%. Their median PFS with ical trial of osimertinib, which enrolled 253 Asian and west- second-line chemotherapy was about four months [79, 80]. ern NSCLC patients with acquired resistance to first- or Because EGFR mutations are detected mostly in patients second-generation EGFR TKIs, as defined by Jackman cri- with an adenocarcinoma or non-squamous histology, the teria [22, 92]. Patients were not preselected according to optimum regimen might be pemetrexed and platinum T790M status [92]. Thirty-one patients were treated across combination treatment [81], followed by maintenance five dose-escalation cohorts (20, 40, 80, 160 and 240 mg oral, pemetrexed for patients who did not suffer from disease daily) and 222 were treated in the dose-expansion cohort. progression [48, 82]. In the dose-escalation cohort, there was no dose-limiting The most common mechanism of acquired resistance toxicity (DLT) and the maximum tolerated dose (MTD) has to EGFR TKIs is acquired T790M mutation. Second- not been reached. Of the 239 evaluable patients, the object- generation EGFR TKIs, including afatinib, dacomitinib ive response rate (ORR) was 51% and the disease control rate and neratinib, had efficacy in inhibiting proliferation of (DCR) was 84%. Patients with EGFR-T790M mutation had a T790M mutation-positive cells in vitro. However, clinical better ORR (61% vs. 21%), DCR (95% vs. 61%), and longer trials showed disappointing results due to high toxicities median PFS (9.6 months vs. 2.8 months) than patients with- resulting from the narrow therapeutic window. In con- out an EGFR-T790M mutation. The drug is relatively safe, trast to second-generation EGFR TKIs, third-generation and most of the AEs were grade 1 and 2. The most common EGFR TKIs had a good treatment effect on tumors har- AEs were diarrhea (47%), skin toxicity (40%), nausea (22%), boring EGFR T790M mutations [48, 83–85]. and anorexia (21%). When patients took higher dose levels (160 and 240 mg), there was an increasing incidence and se- Next-generation (third-generation) epidermal growth factor verity of AEs (rash, dry skin, and diarrhea). Based on efficacy receptor tyrosine kinases inhibitors (EGFR TKIs) and safety, 80 mg daily was selected as the recommended The third-generation EGFR TKIs can form an irreversible dose for further clinical trials [92]. covalent binding to EGFR. They are pyrimidine-based com- Then, a phase II “AURA2” study (NCT02094261) was pounds, and differ from quinazolines-based first-and second- initiated to enroll NSCLC patients with an EGFR-T790M generation EGFR TKIs (Table 2)[86]. Third-generation mutation and acquired resistance to approved EGFR TKIs; EGFR TKIs can attenuate EGFR T790M activity and have the enrollment criteria were similar to those of the AURA Wu and Shih Molecular Cancer (2018) 17:38 Page 7 of 14 Table 2 Different generations of EGFR TKIs Generation EGFR inhibition Drug Molecular Targets Adverse effect Status 1st-generation Reversible; Gefitinib EGFR del19, L858R Skin rash/acne, abnormal LFT FDA approved competitive Erlotinib EGFR del19, L858R FDA approved 2nd-generation Irreversible; covalent Afatinib EGFR del19, L858R, uncommon Diarrhea, paronychia. FDA approved mutations, HER2, HER4 Skin rash Dacomitinib EGFR del19, L858R, HER2, HER4 Diarrhea, skin rash/acne Phase III Neratinib EGFR G719X, HER2, HER4 Diarrhea, dyspnea, N/V Phase II 3rd-generation Irreversible; Osimertinib EGFR mutations and T790M Diarrhea, skin rash FDA approved covalent Rociletinib EGFR T790M mutation, IGF-1R Hyperglycemia, QTc prolong Withdrawn Olmutinib EGFR T790M mutation Diarrhea, skin exfoliation, Approved in South Korea nausea ASP8273 EGFR L858R, del19, T790M, Diarrhea, N/V, Phase III Discontinued thrombocytopenia Nazartinib EGFR L858R, del19, T790M, Rash, diarrhea, pruritus Phase I/II Avitinib (AC0010) EGFR L858R, del19, T790M, Diarrhea, skin rash, Phase I/II abnormal LFT HS-10296 EGFR sensitive mutations None reported Phase I/II (G719X, del19, L858R, L861Q) +/− T790M PF-06747775 EGFR L858R, del19, T790M, None reported Phase I/II N/V nausea and/or vomiting, LFT liver function test, del19 deletion in exon19, EGFR epidermal growth factor receptor, FDA Food and Drug Administration The targets included FDA approved or associated targets study extension cohort. A preplanned pooled analysis was therapy. The investigator-assessed PFS (primary endpoint) performed, including 201 patients from the 80 mg osimer- was significantly longer in the osimertinib arm than in the tinib expansion cohort of AURA and 210 patients from chemotherapy arm (median 10.1 vs. 4.4 months; HR 0.30; AURA2; ORR was 66%, DCR was 91%, and median PFS p < 0.001). The FDA has granted regular approval to the was 11.0 months [93]. third-generation EGFR TKI, osimertinib, for the treatment In the phase III AURA3 study, 419 patients were ran- of patients with metastatic EGFR T790M mutation-positive domized into osimertinib or platinum-pemetrexed chemo- NSCLC. therapy (maintenance pemetrexed was allowed) groups In the preclinical study, osimertinib demonstrated after they had acquired resistance to first-line EGFR TKI greater penetration of the mouse blood-brain barrier than Table 3 Efficacy of third-generation EGFR TKIs in EGFR T790M-positive NSCLC patients Drug Trial Patients (N) Dose ORR T790M PFS (mo.) Osimertinib AURA phase I [92] Total: 253 T790 20-240 mg QD T790M(+): T790M(+): M(+): 138 61% T790M(−): 21% 9.6 T790M(−): 2.8 AURA phase I T790M(+) 63 80 mg QD 71% 9.7 AURA phase II 210 80 mg QD 70% 9.9 AURA phase II extension [132] 411 80 mg QD 62% 12.3 AURA phase III [84] 416 -Osimertinib 71% 10.1 arm: 279 31% 4.4 -Chemotherapy Odds ratio:5.39 HR: 0.30 arm: 140 (95% CI: 3.47–8.48) (95% CI: 0.23–0.41) Rociletinib TIGER-X phase I/II [98] Total: 69 500, 625 or 45% T790M(+): 9.6 T790M(+): 51 750 mg bid T790M(−): 2.8 Olmutinib HM-EMSI-101 phase 76 800 mg QD 62% 6.9 I/II T790M(+) [133] ASP8273 NCT02113813 phase I/II [134] Total: 63 300 mg QD 29% 6.8 T790M(+): 58 Nazartinib NCT02108964 phase I/II [105] 152 75-350 mg QD 46.9% 9.7 Avitinib (AC0010) NCT02330367 phase I/II [106] 136 50-350 mg QD 44% Wu and Shih Molecular Cancer (2018) 17:38 Page 8 of 14 gefitinib, rociletinib, or afatinib [94]. There were several resistance to osimertinib and rociletinib [103]. A multi- reports of dramatic intracranial response to osimertinib in cohort, phase 1 study (NCT02113813) was initiated to evalu- patients with EGFR T790M lung cancer [94, 95]. A phase ate the safety and efficacy of ASP8273 in NSCLC patients I study (BLOOM, NCT02228369), which has enrolled pre- with disease progression after EGFR TKI treatment. The treated EGFR-mutant NSCLC patients with leptomenin- most common AEs included diarrhea (47%), nausea (42%), geal metastasis treated with 160 mg osimertinib once and fatigue (32%). The most common grade 3/4 AE was daily, is ongoing. The preliminary data is promising [96]. hyponatremia (17%). Across all doses, the ORR was 30.7%, and median PFS was 6.8 months in patients with EGFR Rociletinib (CO-1686) T790M [104]. A phase III randomized clinical trial (SOLAR) was conducted to compare the efficacy and safety of Rociletinib, a 2,4-disubstituted pyrimidine compound, is ASP8273 with that of erlotinib or gefitinib as first-line treat- an oral, irreversible, mutant-selective inhibitor of activating ment for advanced EGFR-mutant NSCLC (NCT02588261). EGFR mutations, including T790M, and spares wild-type However, Astellas Pharma (OTCPK: ALPMY) terminated EGFR [97]. TIGER-X (NCT01526928A), a phase I/II trial of the phase III SOLAR study in May 2017 because the rociletinib, enrolled 130 EGFR-mutant NSCLC patients with treatment advantage apparently was not adequate enough to acquired resistance to first- or second-generation EGFR TKIs justify continuation. [83]. The ORR was 59% for the 46 evaluable T790M mutation-positive patients and 29% for the 17 T790M  Nazartinib (EGF816) mutation-negative patients [83]. Because of targeting of IGF- 1R, hyperglycemia (22%) was detected as the most common A phase I/II first-in-human study, NCT02108964 grade 3 AE. An independent updated analysis of the TIGER- (EGF816X2101), investigated nazartinib in EGFR-mutant X trial showed that the T790M mutation-positive patients patients. A total of 152 patients were treated across seven had an ORR of 45% [98]. In addition, a series of cases with cohorts using doses ranging from 75 to 350 mg [105]. response to osimertinib after resistance to rociletinib were Among the 147 evaluable patients, the ORR and DCR reported [99]. Clovis Oncology, Inc. decided to stop enroll- were 46.9% and 87.1%, respectively. The median PFS ment in all ongoing rociletinib studies and terminate the fu- across all dose cohorts was 9.7 months. Skin rash (54%), ture development program in May 2016. diarrhea (37%), and pruritus (34%) were the most com- mon AEs. The skin rashes related to nazartinib were dif- Olmutinib (BI-1482694/HM61713; Olita™) ferent from those caused by other EGFR TKIs in pattern, location, and histology. The most common grade 3/4 AE A phase I/II dose escalation clinical trial, HM-EMSI-101 was diarrhea (16%) [105]. A phase II clinical trial with six (NCT01588145), was initiated in South Korea [100]. Pa- cohorts is ongoing. In addition, a phase Ib/II trial tients took olmutinib in doses ranging from 75 to 1200 mg/ (NCT02335944 and NCT02323126) is ongoing to investi- day. Among the 34 patients with NSCLC harboring T790M gate the efficacy of combined treatments with INC280, a detected by a central laboratory, the ORR was 58.8%. The specific MET inhibitor, and with nivolumab, an anti-PD-1 DCR was 97.1% for patients treated with olmutinib in doses monoclonal antibody in patients with EGFR-T790M mu- greater than 650 mg. The most common DLTs involved tation after acquired resistance to first-line EGFR TKI. gastrointestinal symptoms, abnormal liver function (AST/ ALT), and increasing amylase/lipase levels. Therefore,  AC0010 800 mg/day was selected as the recommended phase II dose. Seventy-six patients with centrally confirmed T790M A phase I/II, first-in-human dose-escalation and expan- mutation-positive NSCLC were enrolled in part II of the sion phase clinical trial (NCT02330367) was carried out study, and 70 were evaluable for response. The ORR was with advanced NSCLC patients with acquired T790M mu- 61% and median PFS was 6.9 months [101]. Based on the tation after first-generation EGFR TKIs treatment [106]. In aforementioned result, olmutinib was first approved in all, 136 patients have been treated across seven cohorts (50, South Korea in 2016. However, Boehringer Ingelheim 100, 150, 200, 250, 300, and 350 mg BID), and MTD has decided to stop the co-development of this drug because of not been reached. The most common drug-related AEs an unexpected grade 3/4 skin toxicity (including palmo- were diarrhea (38%), rash (26%) and ALT/AST elevation. plantar keratoderma) [102]. Grade3/4 AEsof diarrhea(2%), rash (2%) andALT/AST elevation (4%, 2%) were recorded. The 124 evaluable ASP8273 patients had ORR and DCR of 44% and 85%, respectively. Because of the drug safety profile and activity against Preclinical data showed ASP8273 had antitumor activity NSCLC with acquired T790M mutation, a phase II, against EGFR TKI-resistant cells, including those with AEGIS-1 study is ongoing to evaluate treatment efficacy for Wu and Shih Molecular Cancer (2018) 17:38 Page 9 of 14 patients with T790M mutation-positive NSCLC with T790M-positive and T790M-negative tumors was compar- acquired resistance to first-generation EGFR TKIs. An open able (32% vs. 25%; p = .341). The two groups showed no stat- label, randomized phase III trial (NCT03058094) also is on- istical difference in PFS. The NCCN Panel recommends going to compare AC0010 (300 mg, BID) with pemetrexed/ considering an afatinib/cetuximab regimen for patients who cisplatin (4–6 cycles) in patients with advanced NSCLC have progressed after receiving EGFR TKIs and chemother- who have progressed following prior therapy with EGFR apy [48]. However, skin rash (90% all grades) and diarrhea TKI. T790M in biopsy samples was confirmed by a central (71% all grades) were the two most common adverse effects. laboratory. Grades 3 and 4 adverse effects were 44% and 2%, respect- ively. Because of the high rate of AEs with this combination HS-10296 therapy, it is no longer a preferred treatment for patients with tumor harboring EGFR T790M mutations [110]. An open-label, multicenter, phase I/II dose escalation and expansion trial (NCT02981108) is currently recruiting patients  Horizontal pathway with locally advanced or metastatic NSCLC after acquired resistance to first- and/or second-generation EGFR TKIs. Since bypass signaling pathway activation is an important acquired resistance mechanism of EGFR TKIs, it is reason- PF-06747775 able to combine inhibition of EGFR pathway signaling and inhibitors for the bypass signaling pathway to overcome re- PF-06747775 has potent antitumor efficacy against sistance. Different horizontal combination strategies are be- NSCLC harboring a classical mutation with/without ing investigated, but results are preliminary and immature T790M. It significantly attenuates T790M activity and has (Table 4). less toxicity because of the reduction of proteome reactivity MET amplification is an important mechanism of acquired relative to earlier EGFR TKIs [107, 108]. A phase I/II clin- resistance to EGFR TKI therapy [31, 111]. A randomized, ical trial (NCT02349633) involving patients with advanced open-label, phase 2 study enrolled patients with advanced NSCLC harboring EGFR mutations (Del19 or L858R with/ NSCLC (enriched for EGFR-mutant disease) who developed without T790M) is ongoing. acquired resistance to erlotinib to receive emibetuzumab (LY2875358), a humanized IgG4 monoclonal bivalent MET Combination therapy antibody, with or without erlotinib therapy. The ORR of pa- tients whose re-biopsy samples harbored MET overexpres- Vertical pathway sion (≥60%) was 3.8% in the combination arm and 4.8% in the monotherapy arm [112]. In Japan, another phase II Cetuximab is a recombinant human/mouse chimeric clinical trial enrolled 45 patients with advanced EGFR-mu- EGFR IgG1 monoclonal antibody. Combining afatinib and tant NSCLC who developed acquired resistance to first- cetuximab may be useful for patients who have progressed generation EGFR TKIs to receive tivantinib (ARQ197) and after receiving EGFR TKI therapy and chemotherapy [109]. erlotinib combination therapy. The response rate was Among 126 patients, the response rate of patients with 6.7%. High MET expression (≥ 50%) was detected by Table 4 Main mechanisms involved in acquired resistance to EGF receptor-tyrosine kinase inhibitors and the associated targetable drugs Molecular alteration Pathway Targetable drug HER2 amplification Afatinib, Trastuzumab, ado-trastuzumab emtansine (TDM1) MET overexpression/genetic alteration ● Anti-HGF antibody: Rilotumumab, Ficlatuzumab ● Anti-c-MET antibody: MET Mab, Emibetuzumab (LY2875358) ● Selective c-MET inhibitor: Tivantinib (ARQ197), Capmatinib (INC280), Savolitinib (AZD6094), Tepotinib (EMD 1214063), SGX523, SAR125844, ● Multikinase inhibitors: Crizotinib, Cabozantinib (XL184), Glesatinib (MGCD265), Merestinib (LY2801653), S49076 PIK3CA PI3K-AKT-mTOR ● PI3K inhibitor: Pilaralisib (XL147), Dactolisib (BEZ235) and Pictilisib (GDC-0941), Buparlisib (BKM120) ● AKT inhibitor: MK-2206 ● mTOR inhibitor: Everolimus, Temsirolimus, Ridaforolimus BRAF Ras-Raf-MEK-ERK Vemurafenib (PLX4032), Dabrafenib (GSK2118436), Selumetinib, LY3009120 AXL overexpression GAS6-AXL ● Tyrosine kinase inhibitor: Cabozantinib (XL 184) ● AXL antibody: E8, D9, Mab173 ● AXL decoy receptor: AXL-Fc, MYDI Wu and Shih Molecular Cancer (2018) 17:38 Page 10 of 14 immunohistochemical stain in 48.9% of the patients, includ- suppress T-cell function [120]. This finding indicates that ing all three partial responders [113]. In addition, a combin- EGFR functions as an oncogene through cell-autonomous ation of capmatinib (INC280) and gefitinib was tested in a mechanisms and raises the possibility that other oncogenes phase 2 study (NCT01610336) in EGFR-mutant NSCLC may drive immune escape [120]. However, retrospective patients after acquired resistance to gefitinib. EGFR T790M studies showed that NSCLCs harboring EGFR mutations NSCLCs were excluded and high cMET expression was re- were associated with low response rates to PD-1/PD-L1 in- quired. Of the 65 evaluable patients, the ORR was 18% and hibitors, which may have resulted from low rates of concur- DCR was 80%. More responses were seen in tumors with rent PD-L1 expression and CD8(+) TILs within the tumor MET amplifications [114]. microenvironment [119]. A retrospective study on the In addition to MET amplification, different medications are efficacy of nivolumab in patients with EGFR mutation- being investigated to inhibit other bypass signaling pathways, positive NSCLC after EGFR TKI failure found that T790M- including a heat shock protein 90 inhibitor, AUY922 (- negative patients were more likely than T790M-positive ClinicalTrials.gov: NCT01259089 and NCT01646125); a patients to benefit from nivolumab [121]. JAK inhibitor, ruxolitinib (ClinicalTrials.gov: NCT02155465 Different phase 1 trials combining EGFR TKIs with and NCT02145637); a MET/AXL/FGFR inhibitor S- 49076 immunotherapies include nivolumab (ClinicalTrials.gov, (EU Clinical Trials Register:EudraCT Number:2015– number NCT01454102); pembrolizumab (ClinicalTrials.gov, 002646-31) and a PI3K inhibitor, buparlisib (BKM120) number NCT02039674); and atezolizumab (ClinicalTrials.- (ClinicalTrials.gov: NCT01570296 and NCT01487265). gov, number NCT02013219). These studies are all ongoing. Furthermore, combination therapy with osimertinib has been investigated. The TATTON study (ClinicalTrials.gov: Conclusions NCT02143466) enrolled patients who received osimertinib- EGFR TKIs are currently the standard first-line treatment based combination therapy with either a MET inhibitor of patients with advanced NSCLC harboring activating (savolitinib), MEK inhibitor (selumetinib), or anti-PD-L1 EGFR mutations. After acquiring resistance to first-line monoclonal antibody (durvalumab) [115]. However, the EGFR TKI therapy, it is important that the mechanisms of rate of drug-related interstitial disease was high in the osi- acquired resistance in all patients are explored. Then, mertinib plus durvalumab arm, so the development of this based on the mechanism, subsequent treatment can be combination therapy was discontinued [116]. Other clinical chosen. Continuation of EGFR TKI therapy is suitable for trials, including osimertinib in combination with select patients with asymptomatic progression and/or oli- ramucirumab, necitumumab, bevacizumab, or navitoclax goprogression. Repeat tumor biopsy to detect the EGFR (ClinicalTrials.gov, NCT02789345, 02496663, 02803203 T790M mutation is the current standard of care, and osi- and 02520778), are ongoing. mertinib has been approved for patients with acquired Combination therapies have higher rates of toxicities EGFR T790M-mutant disease. Liquid biopsy is an alterna- and side effects than a single agent does. Although the tive method to detect plasma EGFR T790M mutation and aforementioned medications have been evaluated in to identify patients suitable for osimertinib therapy. Com- clinical trials, clinicians should keep in mind the bination therapy may be effective for acquired resistance possibility of AEs when prescribing combination therapy. resulting from activation of the bypass signaling pathway. Advances in the detection method for different resistance Immunotherapy mechanisms and the development of new drugs are both For subsequent therapy, or immunotherapy, nivolumab and urgently needed for personalized therapy. pembrolizumab have been approved as standard treatment, Abbreviations and high-level PD-L1 expression in tumors can predict a 95% CI: 95% confidence interval; AEs: adverse effects; ASCO: American Society of higher response rate. Phase III trials assessing pembrolizu- Clinical Oncology; ctDNA: circulating tumor DNA; DLT: dose-limiting toxicity; mab, nivolumab, or atezolizumab compared to docetaxel as EGFR: epidermal growth factor receptor; EMT: epithelial–mesenchymal transition; ESMO: European Society for Medical Oncology; FDA: Food and Drug Administration; subsequent therapy for patients with metastatic NSCLC MLT: maximum tolerated dose; NCCN: National Comprehensive Cancer Network; found there were no survival benefits for EGFR-mutant NSCLC: non-small cell lung cancer; ORR: objective response rate, DCR disease control lung cancer patients. Also, there were not enough patients rate; OS: overall survival; PD: progressive disease; PFS: progression-free survival; SCLC: small cell lung cancer; TKI: tyrosine kinase inhibitor with these mutations to determine whether there were sta- tistically significant differences. However, immunotherapy Funding was comparable to chemotherapy and was better tolerated. This research did not receive any specific grant from funding agencies in the [117–119]. Until now, there is not enough evidence to public, commercial, or not-for-profit sectors. recommend pembrolizumab, nivolumab, or atezolizumab Authors’ contributions as subsequent therapy for EGFR-mutant patients. SGW and JYS both completed data collection, literature search, generation of In vitro, EGFR-mutant lung cancer cells inhibited antitu- figures, and writing of the manuscript. Both authors read and approved the mor immunity by activating the PD-1/PD-L1 pathway to final manuscript. Wu and Shih Molecular Cancer (2018) 17:38 Page 11 of 14 Competing interests 16. Piotrowska Z, Thress KS, Mooradian M, Heist RS, Azzoli CG, Temel JS, Rizzo C, Jin-Yuan Shih has received speaking honoraria from AstraZeneca, Roche, Pfizer, Nagy RJ, Lanman RB, Gettinger SN, et al. MET amplification (amp) as a Boehringer Ingelheim, Novartis, and Eli Lilly. 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Molecular CancerSpringer Journals

Published: Dec 1, 2018

Keywords: cancer research; oncology

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